1. The application of histone H3K79 methyltransferase inhibitor in improving the development efficiency of animal round sperm injection embryo is characterized in that: the histone H3K79 methyltransferase inhibitor is a histone methyltransferase DOT1L inhibitor.

2. A composition comprising a histone H3K79 methyltransferase inhibitor and a histone H3K9 methyltransferase inhibitor.

3. The composition of claim 2, wherein:

the histone H3K79 methyltransferase inhibitor is a histone methyltransferase DOT1L inhibitor;

the histone H3K9 methyltransferase inhibitor is a histone methyltransferase G9A inhibitor.

4. Use of a histone H3K79 methyltransferase inhibitor or a composition of any of claims 2-3 in the preparation of a product for increasing the efficiency of development of an animal round sperm injected embryo.

5. An activation fluid comprising:

(1) oocyte activating liquid; and

(2) a histone H3K79 methyltransferase inhibitor or a composition according to any one of claims 2 to 3.

6. The activating solution according to claim 5, characterized in that:

the oocyte activating liquid comprises sodium chloride, potassium dihydrogen phosphate, magnesium sulfate, sodium bicarbonate, strontium chloride, bovine serum albumin, disodium ethylene diamine tetraacetate, sodium pyruvate, glutamine, penicillin-streptomycin and sodium lactate.

7. The activating solution according to claim 5 or 6, wherein:

the histone H3K79 methyltransferase inhibitor is a histone methyltransferase DOT1L inhibitor.

8. A culture fluid comprising:

(1) an embryo culture solution; and

(2) a histone H3K79 methyltransferase inhibitor or a composition according to any one of claims 2 to 3.

9. The culture solution according to claim 8, wherein:

the embryo culture solution is a cleavage embryo culture solution;

preferably, the histone H3K79 methyltransferase inhibitor is a histone methyltransferase DOT1L inhibitor.

10. A method for improving the development efficiency of animal round sperm injection embryos comprises the following steps:

placing the oocyte in an activating solution of any one of claims 5 to 7 for pre-activation, and then placing the oocyte in a buffer solution for treatment to obtain a treated oocyte;

placing the round sperm cells into a buffer solution for processing to obtain processed round sperm cells;

injecting the processed round sperm cells into the processed oocytes to obtain round sperm injection embryos;

activating the round sperm injection embryo in the activating solution of any one of claims 5 to 7, and then transferring the round sperm injection embryo into the culture solution of any one of claims 8 to 9 for culture.

Background

Patients with severe azoospermia do not have mature or elongated spermatids in their testis and epididymis, and therefore cannot obtain genetic offspring by Intracytoplasmic sperm injection (ICSI). The Round sperm injection (ROSI) technique is to inject Round sperm into an oocyte, and fertilize the oocyte through auxiliary activation, so as to help the part of azoospermia patients to obtain genetic offspring. Although the ROSI technique has been applied to various animals including mice, rabbits, monkeys, and humans, there are problems such as low implantation rate, high abortion rate, low live productivity, and the like. Therefore, improving the developmental potential of ROSI embryos could strongly drive the clinical application of this technology in the field of assisted reproduction.

Methylation modification of histone H3K79 site plays an important role in the regulation of gene expression, and methylation modification of the site is generally associated with transcriptional activation. The only definite histone methylation modification enzyme at H3K79 site is DOT1L, which can catalyze H3K79 site single/double/trimethyl modification and is used as a histone modification enzyme capable of regulating gene expression. At present, researches on methylation modification functions of H3K79 sites mostly focus on aspects of cell differentiation, DNA damage repair, telomere silencing and the like, and the effect of improving the development efficiency of animal round sperm injection embryos is not reported.

Disclosure of Invention

The first aspect of the invention aims to provide application of a histone H3K79 methyltransferase inhibitor in improving the development efficiency of animal round sperm injection embryos.

In a second aspect, the present invention is directed to a composition.

The third aspect of the invention aims to provide the application of the composition of the second aspect in improving the development efficiency of animal round sperm injection embryos.

The fourth aspect of the invention aims to provide the application of the histone H3K79 methyltransferase inhibitor in preparing products for improving the development efficiency of animal round sperm injection embryos.

The fifth aspect of the invention aims to provide the application of the composition of the second aspect in preparing products for improving the development efficiency of animal round sperm injection embryos.

In a sixth aspect, the present invention provides an activating liquid.

The seventh aspect of the present invention is to provide a culture solution.

The eighth aspect of the invention aims to provide a method for improving the development efficiency of the animal round sperm injection embryo.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

in a first aspect of the invention, the use of a histone H3K79 methyltransferase inhibitor for improving the efficiency of development of animal round sperm injection embryos is provided.

Preferably, the histone H3K79 methyltransferase inhibitor is a histone methyltransferase DOT1L inhibitor.

Preferably, the histone methyltransferase DOT1L inhibitor is SGC 0946.

The molecular formula of SGC0946 is C₂₈H₄₀BrN₇O₄The CAS number is 1561178-17-3, and the chemical structural formula is shown as the formula (I).

Preferably, the final concentration of the histone H3K79 methyltransferase inhibitor is 0.5-1.5 μ M; further 1 to 1.5. mu.M.

Preferably, the embryo development efficiency is at least one of blastocyst development rate, implantation rate and survival rate; further the blastocyst development rate.

Preferably, the animal is a mammal.

In a second aspect of the invention, there is provided a composition comprising a histone H3K79 methyltransferase inhibitor and a histone H3K9 methyltransferase inhibitor.

Preferably, the histone H3K79 methyltransferase inhibitor is a histone methyltransferase DOT1L inhibitor.

Preferably, the histone methyltransferase DOT1L inhibitor is SGC 0946.

The molecular formula of SGC0946 is C₂₈H₄₀BrN₇O₄The CAS number is 1561178-17-3, and the chemical structural formula is shown as the formula (I).

Preferably, the histone H3K9 methyltransferase inhibitor is a histone methyltransferase G9A inhibitor.

Preferably, the histone methyltransferase G9A inhibitor is at least one of a366, BIX-01294, and BRD 4770; further at least one of a366 and BRD 4770; further a 366.

The molecular formula of A366 is C₁₉H₂₇N₃O₂The CAS number is 1527503-11-2, and the chemical structural formula is shown as the formula (II).

The molecular formula of BIX-01294 is C₂₈H₃₈N₆O₂The CAS number is 935693-62-2, and the chemical structural formula is shown as the formula (III).

The chemical formula of the BRD4770 is C₂₅H₂₃N₃O₃The CAS number is 1374601-40-7, and the chemical structural formula is shown as the formula (IV).

Preferably, the final concentration of the histone H3K79 methyltransferase inhibitor is 0.5-1.5 μ M; further 1 to 1.5. mu.M.

Preferably, the final concentration of the histone H3K9 methyltransferase inhibitor is 100-2500 nM; further 150-1250 nM; further, the concentration of the compound is 150 to 300 nM.

In a third aspect of the invention, there is provided the use of a composition of the second aspect for increasing the efficiency of development of an animal round sperm injected embryo.

Preferably, the embryo development efficiency is at least one of blastocyst development rate, implantation rate and live birth rate.

Preferably, the animal is a mammal.

In a fourth aspect of the invention, the application of the histone H3K79 methyltransferase inhibitor in preparing products for improving the development efficiency of animal round sperm injection embryos is provided.

Preferably, the histone H3K79 methyltransferase inhibitor is a histone methyltransferase DOT1L inhibitor.

Preferably, the histone methyltransferase DOT1L inhibitor is SGC 0946.

The molecular formula of SGC0946 is C₂₈H₄₀BrN₇O₄The CAS number is 1561178-17-3, and the chemical structural formula is shown as the formula (I).

Preferably, the final concentration of the histone H3K79 methyltransferase inhibitor is 0.5-1.5 μ M; further 1 to 1.5. mu.M.

Preferably, the embryo development efficiency is at least one of blastocyst development rate, implantation rate and survival rate; further the blastocyst development rate.

Preferably, the product comprises an activation solution and a culture solution.

Preferably, the animal is a mammal.

In a fifth aspect of the invention, there is provided the use of the composition of the second aspect in the manufacture of a product for increasing the efficiency of development of an animal round sperm injected embryo.

Preferably, the embryo development efficiency is at least one of blastocyst development rate, implantation rate and live birth rate.

Preferably, the product comprises an activation solution and a culture solution.

Preferably, the animal is a mammal.

In a sixth aspect of the present invention, there is provided an activating liquid comprising:

(1) oocyte activating liquid; and

(2) a histone H3K79 methyltransferase inhibitor or a composition of the second aspect of the invention.

Preferably, the oocyte activating solution includes sodium chloride, potassium dihydrogen phosphate, magnesium sulfate, sodium bicarbonate, strontium chloride, bovine serum albumin, disodium ethylenediaminetetraacetate, sodium pyruvate, glutamine, penicillin-streptomycin, and sodium lactate.

Preferably, the histone H3K79 methyltransferase inhibitor is a histone methyltransferase DOT1L inhibitor.

Preferably, the histone methyltransferase DOT1L inhibitor is SGC 0946.

The molecular formula of SGC0946 is C₂₈H₄₀BrN₇O₄The CAS number is 1561178-17-3, and the chemical structural formula is shown as the formula (I).

Preferably, the final concentration of the histone H3K79 methyltransferase inhibitor is 0.5-1.5 μ M; further 1 to 1.5. mu.M.

Preferably, the final concentration of histone H3K79 methyltransferase inhibitor in the composition is 0.5-1.5 μ M; further 1 to 1.5. mu.M.

Preferably, the final concentration of histone H3K9 methyltransferase inhibitor in the composition is 100-2500 nM; further 150-1250 nM; further, the concentration of the compound is 150 to 300 nM.

In a seventh aspect of the present invention, there is provided a culture solution comprising:

(1) an embryo culture solution; and

(2) a histone H3K79 methyltransferase inhibitor or a composition of the second aspect of the invention.

Preferably, the embryo culture solution is a cleavage embryo culture solution.

Preferably, the blastomere culture Medium is KSOM culture Medium, M16 culture Medium, CZB culture Medium, G-1TM PLUS (vitrolide, 10128), Sydney IVF Cleavage Medium (Cook, K-SICM) and Quinn' sAt least one of Cleavage Medium (SAGE, ART-1026).

Preferably, the KSOM broth comprises an amino acid additive.

Preferably, the amino acid additive is GlutaMAX^TMAt least one of an additive and a BME amino acid solution.

Preferably, the histone H3K79 methyltransferase inhibitor is a histone methyltransferase DOT1L inhibitor.

Preferably, the histone methyltransferase DOT1L inhibitor is SGC 0946.

The molecular formula of SGC0946 is C₂₈H₄₀BrN₇O₄The CAS number is 1561178-17-3, and the chemical structural formula is shown as the formula (I).

Preferably, the final concentration of the histone H3K79 methyltransferase inhibitor is 0.5-1.5 μ M; further 1 to 1.5. mu.M.

Preferably, the final concentration of histone H3K79 methyltransferase inhibitor in the composition is 0.5-1.5 μ M; further 1 to 1.5. mu.M.

Preferably, the final concentration of histone H3K9 methyltransferase inhibitor in the composition is 100-2500 nM; further 150-1250 nM; further, the concentration of the compound is 150 to 300 nM.

In an eighth aspect of the invention, a method for improving the development efficiency of an animal round sperm injection embryo is provided, which comprises the following steps:

(1) placing the oocyte in the activating solution of the sixth aspect of the invention for pre-activation, and then placing the oocyte in a buffer solution for treatment;

(2) placing round spermatids in a buffer solution for treatment;

(3) injecting the round sperm cell in the step (2) into the oocyte in the step (1) to obtain a round sperm injection embryo;

(4) the round sperm injection embryos are placed in the activating solution of the sixth aspect of the invention for activation, and then transferred into the culture solution of the seventh aspect of the invention for culture.

Preferably, the pre-activation time in the step (1) is 5-15 min.

Preferably, the treatment time in the step (1) and the step (2) is 3-8 min.

Preferably, the buffer solution in the step (1) and the step (2) is M2 culture medium, G-MOPS^TM/G-MOPS^TMPLUS(vitrolife，10130)、ICSI^TM(vitrolife, 10111), Quinn's Advatage medium with HEPES with out Ca, Mg (Sage, ART-4100) and Sydney IVF gauge Buffer (Cook, K-SIGB).

Preferably, the M2 medium, G-MOPS^TM/G-MOPS^TMPLUS(vitrolife，10130)、ICSI^TM(vitrolife, 10111), Quinn's Advatage medium with HEPES with Ca, Mg (Sage, ART-4100) and Sydney IVF gauge Buffer (Cook, K-SIGB) contain cytochalasin B.

Preferably, the activation time in the step (4) is 4-6 h.

Preferably, the culturing time in the step (4) is 12-18 h.

Preferably, the animal is a mammal.

Preferably, the embryo development efficiency is at least one of blastocyst development rate, implantation rate and survival rate; further the blastocyst development rate.

The invention has the beneficial effects that:

the invention discloses the application of a histone H3K79 methyltransferase inhibitor in improving the development efficiency of animal round sperm injection embryos for the first time, and compared with the existing round sperm injection technology, the histone H3K79 methyltransferase inhibitor can obviously improve the blastocyst development rate of round sperm injection embryos, establish a good round sperm injection embryo in-vitro culture system and improve the feasibility of the round sperm injection technology without introducing transgene risk.

The invention also provides a composition which comprises a histone H3K79 methyltransferase inhibitor and a histone H3K9 methyltransferase inhibitor, and the effect of the composition in improving the development efficiency of animal round sperm injection embryos is better than the effect of the two inhibitors used independently. Compared with the prior round sperm injection technology, the composition can obviously improve the blastocyst development rate, implantation rate and survival rate of the animal round sperm injection embryo without introducing the risk of transgenosis; and the round sperm treated by the composition can be injected into live F0 generation to normally breed offspring, so that a good round sperm injection embryo in-vitro culture system is established, and the feasibility of the round sperm injection technology is improved.

Drawings

FIG. 1 is a white light image of the development of round sperm embryos 94h after injection of oocytes with different treatments according to the invention: wherein A is a circular sperm embryonic development white light image after 94 hours of injection of oocytes by circular sperm under DMSO treatment; b is a white light image of the development of round sperm embryos after 94 hours of injection of round sperm into oocytes under the treatment of histone H3K79 methyltransferase inhibitor SGC 0946; c is a white light image of the development of round sperm embryos after 94H of injection of round sperm into oocytes under the treatment of histone H3K79 methyltransferase inhibitor SGC0946 and histone H3K9 methyltransferase inhibitor A366.

FIG. 2 is a graph of blastocyst development rates of round sperm injection embryos treated with various concentrations of histone H3K79 methyltransferase inhibitor SGC0946 in example 2 of the present invention: p < 0.001.

FIG. 3 is a graph of blastocyst development rates for round sperm injected embryos at the time different histone H3K79 methyltransferase inhibitors SGC0946 were used to treat round sperm embryos in example 2 of the present invention: denotes p < 0.01.

FIG. 4 is a graph of blastocyst development rates of round sperm injection embryos treated with different histone H3K79 methyltransferase inhibitors in example 3 of the present invention: p <0.05, p < 0.01.

FIG. 5 is a graph of blastocyst development rates of round sperm injected embryos treated with various concentrations of histone H3K9 methyltransferase inhibitor A366: p < 0.001.

FIG. 6 is a graph of round sperm injection blastocyst development rates at time of round sperm embryo treatment with the different histone H3K9 methyltransferase inhibitor A366: denotes p < 0.01.

FIG. 7 is a graph of blastocyst development rates of round sperm injection embryos treated with different histone H3K9 methyltransferase inhibitors: denotes p < 0.05.

FIG. 8 is a white light map of a 19-day birth mouse after round sperm embryo transfer in example 6 of the present invention: wherein A is a white light image of a mouse born with 19 days of round sperm embryo transplantation under DMSO treatment; b is a white light map of 19-day birth mice treated with histone H3K79 methyltransferase inhibitor SGC0946 and histone H3K9 methyltransferase inhibitor A366 by round sperm embryo transfer.

FIG. 9 is a graph of birth weight and placenta weight of mice born with 19 days of round sperm embryo transfer in example 6 of the present invention: wherein A is the birth weight map of a mouse born by 19 days after round sperm embryo transplantation; b is a graph of the placenta weight of mice born with 19 days of round sperm embryo transfer.

FIG. 10 is a diagram showing the offspring of F0 generation breeding of live mouse injected with round sperm in example 6 of the present invention.

Detailed Description

The present invention will be described in further detail with reference to specific examples.

It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

The reagents used in this example are shown in table 1.

TABLE 1 reagents

The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. The materials, reagents and the like used in the present examples are commercially available reagents and materials unless otherwise specified.

Example 1 Effect of Histone H3K79 methyltransferase inhibitors on the development efficiency of round sperm injected embryos

1. Oocyte collection

(1) Preparing an ovum preservation dish in advance: the cells were cultured in 8 30. mu.L drops of a 3.5cm diameter inlet Eppendorf embryo culture dish in M16 medium, 2.5mL paraffin was added, the culture solution was submerged, and the mixture was placed in an incubator (37 ℃ C., 5% CO)₂) Pre-equilibration for 3 h.

(2) Estrus 10-week-old B6D2F1 females (F1 progeny from mating DBA/2N males with C57BL/6N females, C57BL/6N, DBA/2N purchased from Witongliwa) were selected to weigh approximately 18-25 g. Calculating the unit of hormone injection according to the body weight, and injecting 8U per 20g of body weight; injecting Pregnant Mare Serum Gonadotropin (PMSG) into abdominal cavity at 7 o 'clock or half o' clock at the same day, and injecting Human Chorionic Gonadotropin (HCG) 48h after PMSG is started to induce superovulation; ova were removed 13.5h after HCG injection.

(3) Preheating an M2 culture medium and a hyaluronidase solution to 37 ℃ in advance before egg taking, and putting 100 mu L M2 culture medium into a sterile culture dish for later use; the mice were euthanized by cervical dislocation with the ventral surface facing up, sterilized with 75% alcohol, and then skin was cut along the mid-lower part of the abdomen, torn open to the head and tail, the digestive tract was removed, and the uterine ovary oviduct was exposed. The dilated part of the oviduct which is successfully superexpelled can be seen by naked eyes and is in a transparent vesicular shape, the joint of the oviduct and the uterus is clamped by an ophthalmic forceps, the joint of the ovary and the oviduct is cut off by an ophthalmic scissors close to the ovary, the joint of the oviduct and the uterus is cut off by the ophthalmic forceps close to the ovary, and finally the oviduct is transferred into an M2 culture medium.

(4) The ampulla of the fallopian tube was dissected under the laparoscope with a 1mL syringe needle and oocytes complexes (COCs) were isolated from the ampulla.

(5) Oocyte Complexes (COCs) were transferred to 200. mu.L of M2 medium containing 1mg/mL hyaluronidase, digested for 1 minute on a preheated 37 ℃ scope hot plate during which time 200. mu.L of M2 medium containing 1mg/mL hyaluronidase was digested for 1 minute, with 200. mu.L micropipette to assist with aspiration (note not to blow out bubbles); after 1 minute, most of the granulosa cells around the oocytes are detached; the method comprises the steps of quickly selecting oocytes under a mirror by using a mouth suction tube, respectively cleaning the oocytes in three new 100uL M2 culture medium embryo operation liquid drops for three times, and blowing the oocytes on the upper part of liquid in a circle each time.

(6) And (2) transferring all high-quality MII oocytes (mature oocytes) with obvious edge refraction, uniform and transparent cytoplasm, obvious polar bodies and proper zona pellucida width into the M16 culture medium added with paraffin oil prepared in the step (1) for short-term culture for subsequent microinjection.

2. Round sperm collection

(1) Taking a 10-week B6D2F1 male mouse (F1 filial generation obtained by mating a DBA/2N male mouse and a C57BL/6N female mouse, C57BL/6N, DBA/2N is purchased from Wittingle), separating a testis, weighing 20-25 g, carrying out cervical dislocation euthanasia on the mouse, placing the mouse in an operation tray with the abdomen upward, and spraying 75% ethanol on the abdomen of the mouse for disinfection. The skin is cut off at the horizontal line position of the upper end of the lower limb by scissors, a transverse incision of about 1.5cm is made, then a small incision is made in the muscular layer of the abdominal wall, and the muscular layer is separated bluntly by the scissors, so that the abdominal cavity is exposed. The fat pad of one testis is lightly clamped by blunt forceps, the testis, epididymis and vas deferens are pulled out of the incision together, the testis is separated, and the white membrane of the testis is stripped off and washed for 2 times by PBS.

(2) The testis was transferred to a sterile biosafety cabinet, the fluid was discarded, the tissue was minced with an ophthalmic scissors into a homogenate, 1mL of 0.25% pancreatin was added, the digestion was performed at 37 ℃ in an incubator for 7 minutes, most of the cells were detached from the lumen under the mirror, and 4mL of mouse fibroblast medium (DMEM (12800017, Life Technologies) + 10% FBS (SE200-ES, VISTECH)) was added to terminate the digestion.

(3) The tissue suspension was filtered through a 40 μm cell sieve (Falcon, 352340), transferred to a 15mL centrifuge tube, centrifuged at 348g for 3 minutes, the supernatant was discarded, 4mL mouse fibroblast medium (DMEM (12800017, Life Technologies) + 10% FBS (SE200-ES, VISTECH)) was added to resuspend the cell pellet, Hoechst 33342 (final concentration 2.5 μ g/L) was added, placed in a 37 ℃ water bath in the dark place, stained for 15 minutes, and the mixture was inverted and mixed 3 times during the period to prevent cell adhesion.

(4) After re-centrifugation (3 min at 348 g) the suspension was resuspended in 1mL of flow sort cell stock (PBS + 4% FBS (SE200-ES, VISTECH) and 40 μm cell size was screened into flow tubes to remove adherent cells from the suspension and ensure a single cell suspension before loading.

(5) Haploid, i.e. round sperm, in testicular cells were sorted using a flow cytometric sorter.

3. Round sperm injection

(1) Preparing small molecules and storing: SGC0946 powder (purchased from Target Mol company, CAS: 1561178-17-3, product number: T3082) was dissolved in DMSO at a final concentration of 10mM, stored in a small volume of package at-80 deg.C, and stored for a short period of 4 deg.C to avoid repeated freezing and thawing, and used to prepare an oocyte activator or KSOM culture medium (control group is activator or KSOM culture medium added with DMSO in equal amount) at a final concentration of 1 μ M, wherein the oocyte activator has the following composition: 4.77g/L of sodium chloride, 0.36g/L of potassium chloride, 0.16g/L of potassium dihydrogen phosphate, 0.29g/L of magnesium sulfate heptahydrate, 2.11g/L of sodium bicarbonate, 2.666g/L of strontium chloride hexahydrate, 3.00g/L of bovine serum albumin, 0.041g/L of disodium ethylenediamine tetraacetic acid, 0.03g/L of sodium pyruvate, 0.5% (v/v) of glutamine, 1% (v/v) of penicillin-streptomycin, and 0.45% (v/v) of sodium lactate; the KSOM culture solution contains 1% (v/v) GlutaMAX^TMAdditive, 2% (v/v) BME amino acid solution; it is used as it is.

(2) 15 minutes before injection, the oocytes collected in step 1 are placed into an oocyte activating solution added with SGC0946 for pre-activation, and M2 culture medium containing cytochalasin B (final concentration 5 mug/mL) is placed five minutes before injection to improve the toughness of the cell membranes.

(3) Round sperm cells from flow sorting were free-settled in M2 medium containing cytochalasin B (final concentration 5. mu.g/mL) 5 minutes prior to injection.

(4) Poking the oocyte with a fixed needle to make the pole body at the position of 11-13 points, assisting the injection needle to break the oocyte transparent belt with a large pulse of a piezoelectric rupture instrument (piezo), sucking the round sperm cell with the injection needle, breaking the cell membrane with the suction of the round sperm nucleus, breaking the cell membrane of the oocyte with a small pulse of the piezoelectric rupture instrument (piezo), injecting the round sperm nucleus into the cytoplasm of the oocyte in the MII stage with the injection needle, withdrawing and sucking a small amount of cytoplasm, and sealing the oocyte break.

(5) After the injection is finished, the oocyte is washed three times by the activating solution of the oocyte with the SGC0946 final concentration of 1 μ M prepared in the step (1) to remove cytochalasin B, and is continuously activated for 5 hours, and then the oocyte is transferred into the KSOM culture solution added with the SGC0946 to be CO-cultured for 15 hours (37 ℃, 5% CO)₂) And observing the development condition of the embryo.

(6) And (3) removing the action of small molecules: after 20 hours of co-culture, replacing a new KSOM culture solution with the balanced carbon dioxide concentration for more than 3 hours, and culturing the embryo in the KSOM culture solution with the balanced carbon dioxide concentration for more than 3 hours to a blastocyst at the late stage of the embryo development to 4 cells.

(7) And (3) observing the blastocyst development rate: the blastocyst development rate was calculated 94h after injection.

The results are shown in table 2 and fig. 1: the small molecular compound SGC0946 has an effect of improving the blastocyst development rate of a round sperm injection embryo: the blastocyst development rate is promoted from 33.70% to 53.26%.

TABLE 2 Effect of SGC0946 on embryo development Rate for round sperm injection

Note: a: in the p value <0.05, B6D2F1x B6D2F1 strain, the blastocyst development rate of the SGC0946 group is significantly higher than that of the DMSO group.

Example 2 Effect of concentration and duration of action of Histone H3K79 methyltransferase inhibitors on blastocyst development Rate in round sperm injection embryos

1. Effect of Histone H3K79 methyltransferase inhibitor concentration on blastocyst development Rate of round sperm injection embryos

The procedure is the same as in example 1, steps 1, 2 and 3, except that:

(1) the concentrations of histone H3K79 methyltransferase inhibitor SGC0946 in the activating solution and KSOM culture solution in step 3 of the test process were 500nM, 1. mu.M and 2. mu.M, respectively, and each treatment was repeated 3 times.

The results are shown in FIG. 2: the effect of promoting the development rate of the embryo blastula of the injected round sperms by the histone H3K79 methyltransferase inhibitor SGC0946 with the concentration of 1 mu M is better than that of the histone H3K79 methyltransferase inhibitor SGC0946 with the concentration of 500nM and 2 mu M and the control group (DMSO), and the development rate of the blastula of the injected round sperms is 50.59 percent after the treatment of the histone H3K79 methyltransferase inhibitor SGC0946 with the concentration of 1 mu M.

2. Effect of the time of Histone H3K79 methyltransferase inhibitor treatment of round sperm embryos on the development Rate of round sperm injected embryonal blastula

The procedure is the same as in example 1, steps 1, 2 and 3, except that:

(1) the total time of the histone H3K79 methyltransferase inhibitor-containing oocyte activating solution and KSOM culture solution treatment in step 3 (5) of the test procedure is different: "SGC 09460-10 h" in FIG. 3 indicates that oocytes containing SGC0946 were activated for 5 hours after injection and then co-cultured for 5 hours with KSOM medium; "SGC 09460-20 h" means activation with SGC 0946-containing oocyte activator for 5 hours after injection followed by co-culture with KSOM medium for 15 h; "SGC 09460-44 h" means activation with SGC 0946-containing oocyte activator for 5 hours after injection followed by co-culture with KSOM medium for 39 h; "DMSO" refers to control: after injection, the oocytes were activated for 5 hours with the same volume ratio of the solvent DMSO alone, and then co-cultured for 15 hours with the same volume ratio of the solvent DMSO alone in KSOM medium, and each treatment was repeated 3 times.

The results are shown in FIG. 3: the histone H3K79 methyltransferase inhibitor SGC0946 is treated in 0-20H after the activation of the oocyte injected with the round sperm, and the blastocyst development rate of the round sperm injected embryo is optimal.

Example 3 Effect of Histone H3K79 methyltransferase inhibitors on the development Rate of blastocysts from round sperm injected embryos

The procedure is the same as in example 1, steps 1, 2 and 3, except that:

(1) in the test process, the types and concentrations of the DNA methyltransferases in the chemical activating solution containing the histone H3K79 methyltransferase inhibitor and the KSOM culture solution are different: in fig. 4: "ROSI" means without any treatment; "+ DMSO" indicates the addition of an equivalent amount of DMSO; "SGC 0946-1. mu.M" means SGC0946 at a concentration of 1. mu.M; "EPZ 0047777-1. mu.M" means EPZ0047777(CAS number: 1338466-77-5) at a concentration of 1. mu.M; "EPZ 5676-1. mu.M" means EPZ5676(CAS number: 1380288-87-8) at a concentration of 1. mu.M.

The results are shown in FIG. 4: the histone H3K79 methyltransferase inhibitor SGC0946 can obviously improve the blastocyst development rate of the round sperm injection embryo.

Example 4 Effect of concentration and duration of action of Histone H3K9 methyltransferase inhibitor A366 on blastocyst development Rate in round sperm injection embryos

1. Effect of histone H3K9 methyltransferase inhibitor a366 concentration on blastocyst development rate of round sperm injection embryos the procedure of this experiment was the same as in example 1, steps 1, 2, and 3, except that:

(1) the concentrations of histone H3K9 methyltransferase inhibitor A366 in the activator solution of step 3 and the KSOM culture solution in the test process are 150nM, 300nM and 600nM respectively, and each treatment is repeated 3 times.

The results are shown in FIG. 5: the accelerating effect of the histone H3K9 methyltransferase inhibitor A366 with the concentration of 150nM and 300nM on the embryo blastocyst development rate of the round sperm injection is better than that of the histone methyltransferase inhibitor with the concentration of 600 nM; in particular, the development rate of blastocysts of round sperm injection embryos after the treatment of histone H3K9 methyltransferase inhibitor A366 with the concentration of 300nM is close to 60%.

2. Effect of the time of Histone H3K9 methyltransferase inhibitor A366 treatment of round sperm embryos on the development Rate of round sperm injected embryo blastula

The procedure is the same as in example 1, steps 1, 2 and 3, except that:

(1) the sum of the time spent in the histone H3K9 methyltransferase inhibitor a 366-containing oocyte activator and KSOM broth treatments in step 3 (5) of this experimental procedure was different: in fig. 5: "DMSO" refers to control: after injection, activating the oocyte by using oocyte activating solution only containing DMSO solvent in the same volume ratio for 5 hours, and then co-culturing the oocyte by using KSOM culture solution only containing DMSO solvent in the same volume ratio for 15 hours; "A3660-10 h" means that after injection, the oocyte is activated by using an oocyte activating solution containing A366 for 5 hours, and then is co-cultured by using a KSOM culture solution for 5 hours; "A3660-20 h" means that after injection, the oocyte is activated for 5 hours by using an oocyte activating solution containing A366, and then is co-cultured for 15h by using a KSOM culture solution; "A3660-44 h" means that oocyte activation solution containing A366 is used for 5 hours after injection, and then the oocyte activation solution is used for co-culture for 39 h; each treatment was repeated 3 times.

The results are shown in FIG. 6: the histone H3K9 methyltransferase inhibitor A366 is treated in 0-20H after the activation of the oocyte injected with the round sperm, and the blastocyst development rate of the round sperm injected embryo is optimal.

Example 5 Effect of Histone H3K9 Methyltransferase inhibitor A366 on the development Rate of blastocysts from round sperm injected embryos

The procedure is the same as in example 1, steps 1, 2 and 3, except that:

(1) in the test process, the chemical activating solution containing the histone H3K9 methyltransferase inhibitor and the KSOM culture solution have different types and concentrations of histone methyltransferases: in fig. 7: "DMSO" refers to treatment with an equal amount of DMSO; "A366-300 nM" means A366 at a concentration of 300 nM; "A366-150 nM" means A366 at a concentration of 150 nM; "BIX 01294-200 nM" means BIX-01294(CAS number 935693-62-2) at a concentration of 200 nM; "BIX 01294-100 nM" means BIX-01294 at a concentration of 100 nM; "BRD 4770-2.5. mu.M" means BRD4770 at a concentration of 2.5. mu.M; "BRD 4770-1.25. mu.M" means BRD4770 at a concentration of 1.25. mu.M; "UNC 0631-80 nM" means UNC0631(CAS number 1320288-19-4) at a concentration of 80 nM; "UNC 0631-40 nM" means UNC0631 at a concentration of 40 nM.

The results are shown in FIG. 7: histone H3K9 methyltransferase inhibitor A366, BIX-01294 with concentration of 200nM and BRD4770 all can improve the blastocyst development rate of the injected round sperm embryo.

Example 6 Effect of the combination of Histone H3K79 methyltransferase inhibitor and Histone H3K9 methyltransferase inhibitor on enhancing the development potential of round sperm injected embryos

The procedure is the same as in example 1, steps 1, 2 and 3, except that:

(1) the experimental process also comprises the steps of adopting a DBA/2N male mouse and a C57BL/6N female mouse, wherein the DBA/2N male mouse and the C57BL/6N female mouse are purchased from Beijing Wintotonghua.

(2) In the step 3 activating solution and KSOM culture solution in the test process: 1) the histone H3K9 methyltransferase inhibitor A366 and the histone H3K79 methyltransferase inhibitor SGC0946 are contained at the same time: the final concentrations were 300nM and 1. mu.M, respectively; 2) only histone H3K9 methyltransferase inhibitor a366 was present at a final concentration of 300 nM.

The results are shown in fig. 1 and table 3: the combined use of histone H3K79 methyltransferase inhibitor and histone H3K9 methyltransferase inhibitor can significantly increase blastocyst development rate (66.67%, B6D2F1 strain; 41.18%, DBAxC57 strain), which is better than histone H3K9 methyltransferase inhibitor A366 (58.93%) and histone H3K79 methyltransferase inhibitor SGC0946 (53.26%) alone (B6D2F1 strain).

TABLE 3 Effect of SGC0946 in combination with A366 on blastocyst development Rate of round sperm injected embryos

Note: a: p value <0.05, and the rate of blastula in the A366 group is obviously higher than that in the DMSO group in the B6D2F1xB6D2F1 line; bb, p value <0.01, and the blastocyst rate of the B6D2F1xB6D2F1 group added with SGC0946+ A366 group is obviously higher than that of the DMSO group; the ratio of blastocysts in the DBAxC57 strain with added SGC0946+ A366 group is significantly higher than that in the DMSO group.

(3) The two-cell embryo injected by the strain C57xDBA round sperm is transplanted, and the steps are as follows:

ligation of male mice: taking Kunming male mice with the age of 4-5 weeks, weighing 25-35 g, and purchasing from the experimental animal center of Guangdong province; weighing, injecting 1% sodium pentobarbital (0.8mg/kg) via abdomen for anesthesia, placing the mouse in an operation tray with abdomen facing upwards, and spraying 75% ethanol on the abdomen of the mouse for sterilization; the skin is cut at the horizontal line position of the upper end of the lower limb by scissors, a transverse incision of about 1.5cm is made, then a small incision is made in the muscle layer of the abdominal wall, the muscle layer is separated bluntly by the scissors, and the lower abdominal cavity is exposed. The blunt forceps gently hold the fat pad of one side of the testis, and the fat pad with the testis, the epididymis and the vas deferens are pulled out of the incision together. Vas deferens with blood vessels, peripheral fascia is bluntly separated carefully to avoid damaging peripheral blood vessels, the vas deferens is spread by a pair of forceps, after ligation of operation lines at two ends, the vas deferens at the middle section is burnt and cauterized by high-temperature forceps, and the vas deferens becomes white and hard. After operation, the muscle layer is lifted, the testicular tissue naturally slides to the abdominal cavity, and the steps are repeated to process the vas deferens at the other side. Respectively suturing the abdominal muscle layer and the skin by using medical real silk suture lines; taking estrus female mice to mate with the estrus female mice two weeks after vasectomy, and detecting whether the male mice lose fertility according to whether the female mice can be pregnant.

Preparing a pseudopregnant mother mouse: the pseudopregnant mouse adopts an ICR female mouse, is 8-week-old and 25-35 g in weight, and is purchased from the center of experimental animals in Guangdong province; taking an ICR female mouse in estrus to mate with the ligated male mouse one day before transplantation, detecting the thrombus 8 points before the next day, and if the vaginal opening of the female mouse is detected to have a thrombus block, considering as pseudopregnancy for 0.5 day.

2, embryo oviduct transplantation in cell stage: weighing, injecting 1% sodium pentobarbital (0.8mg/kg) into abdomen for anesthesia, lying on the left side of the mouse, cutting 1cm out of the right side of the center of the back, cutting skin 1cm above thigh, seeing fat pad through muscle layer, separating 0.5cm small opening through muscle layer, clamping fat pad to pull ovary out of abdominal cavity, fixing fat clamp, exposing ampulla, placing a small opening with diameter of 100 μm right above ampulla with 1mL injector under stereoscope, sucking four sections of liquid by oral suction tube, sucking three sections of air in the middle, sucking embryo in the third section of liquid, inserting small opening, blowing embryo in direction of ampulla, transplanting to see three small bubbles gathering in ampulla, transplanting 14 embryos (obtained C57 xBA strain embryo) into one side of mouse, suturing muscle layer and skin, paying attention to no clamping of uterus ovaries, clamping fat pad as much as possible to fix the oviduct, transplanting for 19.5 days, performing cesarean section on a female mouse of a receptor, taking out a fetus and a placenta, weighing, and taking a picture for recording; the results are shown in fig. 8 and table 4: the combination of SGC0946 and A366 can significantly improve implantation rate and survival rate of round sperm injection embryos.

TABLE 4 Effect of SGC0946 in combination with A366 on implantation and live birth rates of round sperm injected embryos

Note: p <0.05, and the implantation rate of round sperm injection embryos incubated by SGC0946 and A366 in DBAXC57 strain is obviously higher than that of DMSO group; p <0.05, circular sperm injection embryo implantation rate in DBAXC57 line incubated with SGC0946 and a366 in combination was significantly higher than in DMSO group.

Birth weight and placenta weight were counted, and the results are shown in fig. 9: in fig. 9: "ROSI + DMSO" means the addition of DMSO; "ROSI + AS" indicates the addition of SGC0946 and A366; n represents the number of repetitions; the birth weight and the placenta weight of the round sperm injection live mice added with SGC0946 and A366 showed no significant difference compared with the DMSO group.

Taking an ICR female mouse naturally produced 0-5 days before cesarean section as a generation female mouse, taking the generation as lactation, breeding a live fetus produced by cesarean section, and continuously observing whether the offspring is fertile or not, wherein the result is shown in figure 10: circular sperms added with SGC0946 and A366 are injected into live mice for F01 generation to normally breed offspring.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.